Numerous bioproducts including primary and secondary metabolites, recombinant proteins, and other biopolymers are produced by microbial fermentation. However, natural organisms are not optimized for this task, and product yield is often limited by NADPH or other metabolites. In order to improve bioproduct production, microorganisms may be engineered to enhance their metabolic capacity for chemicals such as NADPH.
The cofactor pair NADPH/NADP+ plays a central role as donors and/or acceptors of reducing equivalents during anabolic metabolism. The NADH/NAD+ pair alternatively is used primarily for catabolic activities of the cell. Together these cofactors influence virtually every oxidation-reduction metabolic pathway in the cell.
As one example, polyhydroxyalkanoates (PHAs) are a family of biodegradable polyesters synthesized by numerous microorganisms and function as an intracellular carbon and energy storage material. The best characterized PHA is polyhydroxybutyric acid (PHB). PHB is synthesized from acetyl-CoA in three sequential reaction steps catalyzed by the enzymes of the phb operon: β-ketothiolase, acetoacetyl-CoA reductase and PHB synthase, and the acetoacetyl-CoA reductase reaction requires NADPH as a cofactor. Due to the high cost and difficulty regenerating NADPH coenzymes, the enzymatic production of NADPH-dependent compounds such as PHB is challenging.
As a second example, three groups of FAD-dependent monooxygenases that use NADPH as a cofactor include flavin-containing monooxygenases (FAO), N-hydroxylating monooxygenases, and Baeyer-Villiger monooxygenases (BVMO). Enzymes of the BVMO type have been particularly interesting for their ability to form chiral lactone products from substituted cyclohexanones (Willetts, 1997; Stewart, 1998; Stewart, 2000), and an FAO enzyme that produces indigo has also been reported (Choi, 2003). Many BVMO enzymes have been isolated and mechanistic studies, 3-D structure and biochemical studies provides a detailed understanding of their reactions and allows their manipulation to produce desired products. The use of such enzymes in synthetic chemical processes and for the development of new drugs are of considerable importance, but the problem of limiting NADPH must be solved for these “green” resources to be commercially realized.
NADPH is normally generated through the oxidative part of the pentose phosphate pathway by the action of glucose-6-phosphate dehydrogenase (ZWF1):Glucose+NADP+→gluconolactone-6-P+NADPH+H+Verho R, et al. (2003).
However, there are many other ways of generating NADPH. Fructose can be converted to mannitol, oxidizing two molecules of NAD(P)H using mannitol dehydrogenase and erythritol-4-phosphate dehydrogenase. Maicas, et al., (2002). Another protein that could be activated or overexpressed to increase NADP production is glucose dehydrogenase (GDH), per the following reaction:Glucose+NADP+→d-glucolactone+NADPH
Currently, several systems have been used to supply NADPH, including glucose dehydrogenase (Kataoka, 2003; Kizaki, 2001), alcohol dehydrogenase, and NADH-NADPH transhydrogenase. NADH-NADPH transhydrogenase is induced in E. coli strains following exposure to agents such as H2O2, however such methods are not useful due to the deleterious effects of the agents. Therefore, methods of increasing the intracellular levels of the cofactor NADPH are required that do not otherwise damage the cell or detract from its ability to produce large amounts of desirable product.
NADP-Dependent glucose dehydrogenases have been used for both bioproduction and NADPH generation in a continuous manner. Kizaki, et al. (2001) used E. coli cells expressing both the carbonyl reductase (S1) gene from Candida magnoliae and the glucose dehydrogenase (GDH) gene from Bacillus megaterium to synthesize enantiomeric ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) from ethyl 4-chloro-3-oxobutanoate (COBE). Liu, et al. (2005) have used a 2 cell system to perform a generation of ethyl (R)-4-chloro-3-hydroxybutanoate, but cell extracts were used in a non-renewing system with one cell expressing an NADPH-dependent aldehyde reductase gene for the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate and a second cell expressing glucose dehydrogenase gene for NADPH generation. These systems demonstrate the benefits of bacterial NADPH production and use of NADPH dependent enzymes to generate relevant compounds. But stoichiometric amounts of cofactor are required and these systems lack a continuous, high level production of NADPH that is required to allow these reactions to proceed.
The present invention provides methods to increase intracellular availability of NADPH, allowing the increase of products that require this cofactor in their biosynthesis pathways. An example is the production of polyhydroxybutyrate. Other applications are the production of amino acids, lycopene, lactones, chiral alcohols, pharmaceutical intermediates and other biosynthetic products. The system will work with a variety of carbon source feed stocks for applications from bioproduction to bioremediation.